A typical lager sweet wort consists of a complex mixture of starch derived carbohydrates, which are classified as fermentable or non-fermentable according to whether they can be converted into ethanol by brewer's yeast. The fermentable carbohydrates are formed by hydrolysis of grain starches by two enzymes, .alpha. and .beta. amylase, derived from malted barley. In most American lagers malted barley also serves as the predominant starch source while a smaller proportion is derived from nondiastatic adjunct grains. In the United States, corn grits and #4 brewer's rice are the predominant adjuncts.
All grain starches are glucose homopolymers in which the glucose residues are linked by either .alpha.-1,4 or .alpha.-1,6 bonds. During the mashing cycle the starches are first solubilized and then a portion of the solubilized large starch molecules are hydrolyzed to three low molecular weight sugars which brewer's yeast can ferment to ethyl alcohol. The major fermentable sugars are glucose, maltose, and maltotriose while traces of sucrose and fructose are also present. The nonfermentable or limit dextrin fraction consists of all sugars of a higher degree of polymerization (DP) than maltotriose. The bulk of the limit dextrin fraction is composed of polysaccharides which are greater than 10,000 molecular weight.
As indicated above the hydrolysis of the grain starches is catalyzed by two amylases endogenous to malted barley. One, .alpha.-amylase, is an endoamylase which randomly cleaves .alpha.-1,4 bonds in the interior of the raw, largely insoluble starch molecules, fragmenting it into large but soluble polysaccharides termed dextrins. The second .beta. amylase is an exo-amylase which sequentially cleaves .alpha.-1,4 bonds from the non-reducing end of these dextrins producing the three fermentable sugars described above. Both enzymes are inactive towards the .alpha.-1,6 linkages (branch points) of the starches (i.e. they are unsable to debranch the strach molecule) and this results in the formation of the limit dextrins described above.
After completing the mash cycle, the spent grains are removed by passing the mash through a lauter tun to obtain the clarified lager sweet wort. The wort is then transferred to a brew kettle and boiled vigorously for 1-2 hours to inactivate the malt enzymes. It is then cooled, pitched with yeast, and fermented at temperatures ranged from 8.degree.-16.degree. C. to convert the three sugars described above to ethanol. The composition of the wort can vary depending on the starting materials, mash cycle, and other variables. The carbohydrate composition of a typical wort consists of 65-80% fermentable sugars, and limit dextrins ranging from 20-35%. At end of fermentation the fermentable fraction would be converted to ethanol at a final concentration ranging from 3-6% w/w. The limit dextrins are not converted during fermentation and form the bulk of the dissolved solids, commonly referred to as real extract, in the final beer.
Recently, reduced calorie beers have become popular in the U.S. beer market. These beers may be formulated by: (1) reducing both the alcohol and real extract concentrations in the beer to attain the desired calorie level, or (2) by hydrolyzing the limit dextrins with exogenous enzymes, one component of which is capable of debranching the limit dextrins. The latter method is advantageous since it allows one to attain the desired calorie level with a minimum reduction of the alcohol content of the packaged product. The enzyme most commonly used to hydrolyze the limit dextrins is glucoamylase, a nonspecific exoamylase derived from a variety of fungal sources (e.g. A. niger, R. delmar, etc.) [1]. The enzyme is active vs. both .alpha.-1,4 and .alpha.-1,6 linkages and therefore is capable of completely hydrolyzing starch to glucose. It attacks the starch molecule from the nonreducing end producing glucose as the sole end product. It is also active vs. starch derived oligosaccharides, e.g. maltose, maltotriose, isomaltose, etc.
In theory debranching enzymes may be added at any time during the brewing process. In practice brewers prefer to add them in fermentation because the fermentation process itself requires 6-15 days depending on pitching rate, fermentation, temperature, etc. In contrast the brewhouse operations are of much shorter duration (2-4 hrs/brew) and it operates under tight scheduling constraints. Therefore these enzymes are employed as fermentation adjuncts as taught by Gablinger in U.S. Pat. No. 3,379,534, and the limit dextrins are hydrolyzed to fermentable sugars, which the yeast convert to ethanol. Operationally these beers ferment to a lower specific gravity due to: (1) increased alcohol, and (2) decreased real extract, than would the same beer without exogenous enzymes. Such beers are referred to as superattenuated beers. [2].
The exogenous enzymes described above are dissolved in the wort or beer. It would be economically adventageous to have a method of enzymatically treating beer without dissolving the enzymes in the beer. One method is to immobilize the enzyme on a water insoluble solid support or carrier in such a manner that: (1) the immobilized enzyme can be readily recovered and reused and (2) the system is stable to repeated use for a prolonged period of time.
Enzymes have been immobilized by adsorption, entrapment, and covalent attachment to a wide series of supports. Briefly, adsorption relies on electrostatic or van der Waals type bonds for attachment of the enzyme to the solid support. Thus, many enzymes have been adsorbed on various ion exchange resins, clays, etc. The entrapment method entails polymer formation from a solution containing the enzyme. The enzyme is then physically entrapped in the interstices of the polymer matrix as it is formed and remains there due to the fact that the enzyme is too large to diffuse from the matrix back into solution. This technique is frequently used for conversion of low molecular weight substrates which can diffuse into the matrix and contact the enzyme. Obviously, it cannot be used for conversion of macromolecular substrates since the very matrix entrapping the enzyme would not permit entry of large substrates and would thus prevent enzyme-substrate contact.
The final method involves covalent attachment of the protein molecule to a polymer bearing a reactive functional group. The enzyme may be attached via one of its free functional group (i.e., groups not involved in the peptide linkage). These include: (1) the amino group of lysine; (2) the phenolic ring of tryosine; (3) the .omega. COOH group of aspartic or glutamic acids; and (4) the imidazole ring of histidine. Another potentially reactive group is the carbohydrate moiety of glycoproteins. Theoretically covalent attachment should provide the most stable immobilized adducts.
The main reasons for immobilizing enzymes on water insoluble supports are: (1) recovery and reuse of the enzyme, (2) preparation of a relatively enzyme-free product, (3) to fashion the immobilized derivative into a reactor through which the substrate stream may be rapidly circulated and still effect conversion to the desired product and most importantly (4) to fashion an operationally stable system; i.e. one in which the immobilized derivative maintains its catalytic potency under the defined operating conditions for a large number of cycles over a long period of time.
Several types of immobilized enzyme reactors have been described in the literature [3]. The most prevalent are those in which the enzymes have been immobilized on particulate carriers. These include: (1) batch stir in which the immobilized adduct is stirred in the substrate stream and recovered by filtration, (2) plug flow or fixed bed in which the immobilized derivative is packed into a column and the substrate stream is passed through it in a manner similar to a column chromatography operation, and (3) fluidized bed, which is similar to plug flow except that the substrate stream is circulated into the bottom of the column at sufficiently greater flow rates to float or fluidize the bed.
Reactors have also been constructed from enzymes which have been immobilized on various membranes. Thus several enyzmes have been immobilized on collagen films, which were then fashioned into concentric cylindrical reactors [4, 6].
The reactor types fashioned from enzymes immobilized on particulate carriers are not suitable for processing a beer during primary fermentation. First of all, a fermenting beer stream (like many other industrial substrate streams) contains a large concentration of suspended solids formed or introduced as follows. When the wort is cooled after kettle boil, a heavy precipitate forms which is allowed to settle out in a tank. The precipitate (a mixture of protein, carbohydrate, etc.) is referred to as trub and the settling process is referred to as hot-break. Trub separation is not complete during the hot-break, and its formation continues even during fermentation. In addition, beer is pitched with a large (on the order of 1.times.10.sup.7 cells/ml of wort) concentration of brewer's yeast at the beginning of the fermentation. The yeast typically multiplies to six to nine times its original concentration at high kraeusen and then settles out as the specific gravity decreases toward the end of fermentation.
In addition to trub formation and yeast multiplication there are two other major changes that occur during fermentation: (1) large quantities of CO.sub.2 are evolved during active fermentation, and (2) the specific gravity of the beer decreases markedly throughout.
Finally, it is economically necessary for brewers to reclaim most of the expanded yeast crop at end fermentation to repitch fresh wort in order to supplement the crop produced by primary propagation. Typically brewers are able to pitch 3-6 fresh fermentations with the yeast reclaimed from one fermenter.
With these facts in mind, it becomes clear why the three particulate reactors described above are not suited to this stream:
(1) The batch stir system is impractical since it would require that the yeast and the immobilized derivative be separated from each other at end of fermentation in order to recover and reuse both the enzyme conjugate and the yeast cream. This separation would prove both difficult and costly.
(2) The plug flow reactor could not support flow of a substrate stream containing large and variable levels of suspended solids typical of a fermenting lager stream. Such a system would rapidly plug as the solids (yeast and trub) accumulated on top of the immobilized enzyme bed.
(3) Fluidized beds are impractical since the density of the fermenting beer stream is continuously decreasing during fermentation. Since particle flotation is dependent on the density of the supporting medium, the flow rate would have to be continuously increased throughout fermentation to compensate for the density decrease in order to keep the bed fluidized. In addition, during active fermentation the large quantities of CO.sub.2 evolved would disrupt the even flow of liquid and make fluidization more difficult and possibly channel the bed. Most fluidized bed reactors contain support retainers at both ends in order to prevent flow of the adduct back into the substrate feed tank or into the product receiver. The suspended solids could accumulate at the retainers and block flow. Finally at end of fermentation the adduct would have to be separated from the yeast that would be entrained with the residual beer.
Membrane reactors of the type described in the literature [4, 5] suffer from the fact that the membranes lack strength and require backing on large amounts of inert support materials.
Immobilized glucoamylase reactors have been applied to the production of high glucose syrups from liquefied corn starch. These substrates are readily soluble in water at concentrations up to 40% w/w and present none of the problems attendant to a fermenting beer stream as described above. Thus most conventional plug flow reactors are able to handle these substrate streams. British Pat. No. 1,421,955 by Woodward and Bennett discloses the use of a glucoamylase immobilized on a particulate carrier to: (1) sweeten an ale or stout post fermentation by contacting the end fermented beverage with immobilized glucoamylase (GA) in order to hydrolyze the limit dextrins to glucose, and (2) to convert clarified sweet wort limit dextrins to glucose prior to fermentation. They also state that a fluidized bed reactor fashioned from this carrier could work during fermentation, but the patent describes no such use of a fluidized bed reactor. Further such a reactor is impractical in a production situation for the reasons discussed above, i.e. rapidly changing fluid density, plugging at retainers, CO.sub.2 evolution, and necessity of separating the conjugate from the yeast.
Most commercial glucoamylase is isolated from the mold Aspergillus niger. The glucoamylase produced by this microorganism is extracellular. The enzyme is a glycoprotein containing approximately 16% carbohydrate [7]. It is known that the hexose residues of the sugar moiety of glycoproteins may be oxidized by periodic acid to yield a protein containing reactive aldehydic functional groups. The resulting aldehydes may then be reacted with supports possessing primary amines in aqueous media under mild conditions to form the aldiminine or Schiff's base derivatives (Zaborsky U.S. Pat. No. 3,970,521).